The present invention relates generally to methods and compositions useful for immobilizing anti-ligands on surfaces. The immobilized anti-ligands, which can be, for example, hormones or hormone receptors, antibodies or antigens, oligosaccharides, and oligonucleotides, may be used in a variety of screening and assay methodologies for ligands in liquid media.
Certain biological molecules are known to interact and bind to other molecules in a very specific manner. Essentially any molecules having a high binding specificity for each other can be considered a ligand/anti-ligand pair, e.g., a vitamin binding to a protein, a cell-surface receptor binding to a hormone or drug, a glycoprotein serving to identify a particular cell to its neighbors, an IgG-class antibody binding to an antigenic determinant, an oligonucleotide sequence binding to its complementary fragment of RNA or DNA, and the like.
The specific binding properties of anti-ligands for ligands have implications for many fields. For example, the strong binding affinity of antibodies for specific determinants on antigens is critical to the field of immunodiagnostics. Additionally, pharmaceutical drug discovery, in many cases, involves discovering novel drugs having desirable patterns of specificity for naturally-occurring receptors or other biologically important anti-ligands. Many other areas of research exist in which the selective interaction of anti-ligands for ligands is important and are readily apparent to those skilled in the art.
The immobilization of anti-ligands onto surfaces is an important step in performing repetitive assays and screenings of ligands with solid phase systems. Previous methods of attaching anti-ligands to surfaces are limited by low reaction efficiencies or by a general inability to regionally and selectively attach a plurality of anti-ligands to the surface.
A large variety of methods are known for attaching biological molecules to solid supports. See generally, Affinity Techniques. Enzyme Purification: Part B. Methods in Enzymology, Vol. 34, ed. W. B. Jakoby, M. Wilchek, Acad. Press, N.Y. (1974) and Immobilized Biochemicals and Affinity Chromatography, Advances in Experimental Medicine and Biology, vol. 42, ed. R. Dunlap, Plenum Press, N.Y. (1974), which are incorporated herein by reference. For example, U.S. Pat. No. 4,681,870 describes a method for introducing free amino or carboxyl groups onto a silica matrix. These groups may subsequently be covalently linked to, e.g., a protein or other anti-ligand, in the presence of a carbodiimide. Alternatively, a silica matrix may be activated by treatment with a cyanogen halide under alkaline conditions. The anti-ligand is covalently attached to the surface upon addition to the activated surface. Another example is presented in U.S. Pat. No. 4,282,287, which describes a method for modifying a polymer surface through the successive application of multiple layers of biotin, avidin and extenders. Also, U.S. Pat. No. 4,762,881 describes a method for attaching a polypeptide chain to a solid substrate by incorporating a light-sensitive unnatural amino acid group into the polypeptide chain and exposing the product to low-energy ultraviolet light.
Similarly, a variety of techniques have been developed for attaching oligonucleotides to surfaces. For example, U.S. Pat. No. 4,542,102 describes a method employing a photochemically active reagent (e.g., a psoralen compound) and a coupling agent, which attaches the photoreagent to the substrate. Photoactivation of the photoreagent binds a nucleic acid sequence to the substrate to give a surface-bound probe for a complementary oligonucleotide of the sequence. However, this method has low quantum yields in protic solvents, lacks spatial directability, and relies upon initial affinity between the photoreagent and nucleic acids prior to photoactivation.
U.S. Pat. No. 4,562,157 describes a technique for attaching biochemical ligands to surfaces by attachment of a photochemically reactive aryl azide. Irradiation of the azide creates a reactive nitrene which reacts irreversibly with macromolecules in solution resulting in the formation of a covalent bond. The high reactivity of the nitrene intermediate, however, results in both low coupling efficiencies and many potentially unwanted products due to nonspecific reactions.
Thus, there exists a need for improved methods for attaching a broad range of anti-ligands to predefined regions of a solid support surface. The methods should efficiently provide stable attachment of selected anti-ligands to the activated surface regions, yet attachment should be restricted to the activated regions. The present invention fulfills these and other needs.